Methods and kits for measuring von willebrand factor

ABSTRACT

Methods and kits for measuring levels of von Willebrand factor function in a sample without using a platelet aggregation agonist, such as ristocetin, comprising recombinant glycoprotein Ibα having a combination of G233V, D235Y and M239V mutations and an agent to detect a complex between the recombinant glycoprotein Ibα and von Willebrand factor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/669,866, filed Nov. 6, 2012 (now issued as U.S. Pat. No. 8,865,415),which is a continuation of U.S. patent application Ser. No. 13/153,105,filed Jun. 3, 2011 (now issued as U.S. Pat. No. 8,318,444), which is acontinuation of U.S. patent application Ser. No. 12/197,057, filed Aug.22, 2008 (now issued as U.S. Pat. No. 8,163,496), which claims thebenefit of U.S. Provisional Patent Application No. 60/957,604, filedAug. 23, 2007, all of which are incorporated by reference as if setforth in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with United States government support underRO1-HL033721-19 and RO1-HL081588-03, awarded by the National Institutesof Health. The government has certain rights in this invention.

BACKGROUND

The invention relates generally to methods and kits for measuring vonWillebrand factor (VWF), and more particularly to methods and kits formeasuring VWF that do not require a platelet aggregation agonist, suchas ristocetin.

VWF is a multimeric glycoprotein synthesized by megakaryocytes andendothelial cells, which is subsequently secreted into blood plasma as aspectrum of multimers. VWF binds other proteins, especially proteinsinvolved in hemostasis, such as Factor VIII (an essential clottingfactor that participates in the intrinsic pathway of blood coagulation)and platelet glycoprotein Ib (GPIb; a component of a platelet adhesionreceptor complex). VWF is deficient or defective in von Willebranddisease (VWD) and is involved in a large number of other diseases,including thrombotic thrombocytopenic purpura, Heyde's syndrome andpossibly hemolytic-uremic syndrome. See, Sadler J, “Biochemistry andgenetics of von Willebrand factor”. Annu Rev. Biochem. 67:395-424(1998). VWF levels can be affected by many factors including ABO bloodgroup and ethnicity.

VWD is a common bleeding disorder characterized by either qualitative orquantitative defects in tests for VWF. Symptoms of VWD include easybruising, menorrhagia and epistaxis. Currently, many types of hereditaryVWD are known (e.g., type 1; type 2A, 2B, 2M, 2N and type 3, as well asplatelet-type, pseudo VWD, which results from a defect in plateletGPIb); however, acquired forms of VWD are also known, but are lessfrequently observed. Of particular interest herein is platelet-type,pseudo VWD. In contrast to the other forms of VWD, the genetic defect inplatelet-type, pseudo VWD is in platelets rather than VWF and ischaracterized by abnormally high binding affinity of an individual'splatelets to VWF, leading to a characteristic platelethyper-responsiveness in vitro to a low concentration of ristocetin.

Additional screening tests for VWD include those that measure FactorVIII activity, VWF antigen (VWF:Ag), VWF binding to collagen (VWF:CB)and VWF ristocetin cofactor activity (VWF:RCo). Of particular interestherein is VWF:RCo, which is presently the standard for measurement ofVWF function. VWF:RCo utilizes an ability of VWF to bind platelet GPIbfollowing activation by ristocetin, which results in a VWF-dependentagglutination of platelets that can be measured quantitatively byplatelet aggregometry or turbidometry. See, Macfarlane D, et al., “Amethod for assaying von Willebrand factor (ristocetin cofactor),”Thromb. Diath. Haemorrh. 34:306-308 (1975). In fact, an internationalreference standard for VWF:RCo was assigned a biologic activity ininternational units by the World Health Organization (WHO) and theScientific and Standardization Committee of the International Society onThrombosis and Haemostasis (ISTH).

Unfortunately, VWF:RCo, has several shortcomings. For one, VWF:RCo hashigh intra- and inter-assay imprecision because of its dependence onristocetin. See, e.g., Chng W, et al., “Differential effect of the ABOblood group on von Willebrand factor collagen binding activity andristocetin cofactor assay,” Blood Coagul. Fibrinolysis 16:75-78 (2005);Favaloro E, “An update on the von Willebrand factor collagen bindingassay: 21 years of age and beyond adolescence but not yet a matureadult,” Semin. Thromb. Hemost. 33:727-744 (2007); and Riddel A, et al.,“Use of the collagen-binding assay for von Willebrand factor in theanalysis of type 2M von Willebrand disease: a comparison with theristocetin cofactor assay,” Br. J. Haematol. 116:187-192 (2002).Federici et at recently described an alternative assay with improvedreproducibility that used recombinant GPIb in an enzyme-linkedimmunosorbant assay of VWF binding; however, it is ristocetin dependent.See, Federici A, et al., “A sensitive ristocetin co-factor activityassay with recombinant glycoprotein Ib for diagnosis of patients withlow von Willebrand factor levels,” Haematologica 89:77-85 (2004).

In addition, VWF:RCo does not always reflect the true in vivo functionof VWF when mutations or polymorphisms are in the ristocetin-bindingregion of VWF. For example, some individuals have VWF mutations thatshow a reduced interaction with ristocetin such that VWF:RCo is markedlyreduced (e.g., <0.12 IU/dL), although they have no bleeding symptomseven with a major surgical challenge. See, Flood V, et al., “Common VWFhaplotypes in normal African-Americans and Caucasians recruited into theZPMCB-VWD and their impact on VWF laboratory testing,” Blood 10:Abstract714 (2007); Mackie I, et al., “Ristocetin-induced platelet agglutinationin Afro-Caribbean and Caucasian people,” Br. J. Haematol. 50:171-173(1982); and Miller C, et al., “Measurement of von Willebrand factoractivity: relative effects of ABO blood type and race,” J. Thromb.Haemost. 1:2191-2197 (2003). These individuals, who appear to have apolymorphism in the ristocetin-binding region, do not have anabnormality in the binding of VWF to platelet GPIb.

Furthermore, VWF:RCo is affected by high-affinity VWF/plateletdisorders. For example, individuals with platelet-type, pseudo VWD haveGPIb mutations that cause spontaneous binding of their platelets to VWF.See, Franchini M, et al., “Clinical, laboratory and therapeutic aspectsof platelet-type von Willebrand disease,” Int. J. Lab. Hematol. 30:91-94(2008); Miller J & Castella A, “Platelet-type von Willebrand's disease:characterization of a new bleeding disorder,” Blood 60:790-794 (1982);and Miller J, “Platelet-type von Willebrand's Disease,” Thromb. Haemost.75:865-869 (1996). Likewise, individuals with type 2B VWD have VWFmutations that cause spontaneous binding to platelets. See, Weiss H,“Type 2B von Willebrand disease and related disorders of patients withincreased ristocetin-induced platelet aggregation: what they tell usabout the role of von Willebrand factor in hemostasis,” J. Thromb.Haemost. 2:2055-2056 (2004).

Because of the wide variability and reproducibility of VWF:RCo, the artdesires a VWF function assay that does not require a plateletaggregation agonist, such as ristocetin (i.e., ristocetinless).

BRIEF SUMMARY

The invention relates generally to methods and kits for measuring VWFwithout requiring a platelet agglutination agonist by utilizingrecombinant platelet GPIb gain-of-function mutations. As used herein, a“platelet agglutination agonist” means an agent that facilitatesadhesion between VWF and GPIb in platelet agglutination tests. Examplesof platelet agglutination agonist include, but are not limited to,ristocetin and botrocetin.

In a first aspect, the present invention is summarized as a method ofmeasuring VWF without requiring a platelet agglutination agonist byproviding a surface with immobilized recombinant platelet GPIbα havingat least two mutations selected from G233V, D235Y and M239V relative toSEQ ID NO:2 or a functional fragment thereof. The method also includescontacting a sample having or suspected of having VWF with theimmobilized GPIbα or functional fragment thereof without using theplatelet agglutination agonist. The method also includes detecting acomplex, if any, of VWF and the immobilized GPIbα or functional fragmentthereof.

In some embodiments of the first aspect, the surface can be a cellsurface such that the method is a flow cytometry (FC) orfluorescence-activated cell sorting (FACS) assay. Suitable host cellscan be a Xenopus oocyte, CHO-K1 cell, L929 cell, HEK-293T cell, COS-7cell or S2 cell engineered to comprise a polynucleotide encodingplatelet GPIbα having the at least two mutations selected from the groupconsisting of G233V, D235Y and M239V relative to SEQ ID NO:2 or afunctional fragment thereof. The host cell also can be engineered tofurther comprise a polynucleotide encoding platelet glycoprotein Ibβ(GPIbβ; SEQ ID NO:4) and/or optionally platelet glycoprotein IX (GP-IX;SEQ ID NO:8) or functional fragments thereof.

In some embodiments of the first aspect, the surface can be asolid-phase surface such that the method is an enzyme-linkedimmunosorbant assay (ELISA). The solid-phase surface can be agarose,glass, latex or plastic.

In some embodiments of the first aspect, the complex can be detectedwith a labeled anti-VWF antibody or functional fragment thereof, such asa fluorescently labeled antibody or fluorescently labeled functionalfragment thereof. Alternatively, the complex can be detected by surfaceplasmon resonance or quasi-elastic light scattering.

In some embodiments of the first aspect, the sample can be a biologicalsample from an individual having or suspected of having VWD, such asplasma.

In some embodiments of the first aspect, the at least two mutations canbe D235Y/G233V, D235Y/M239V or G233V/M239V. In other embodiments of thefirst aspect, the at least two mutation can be a triple mutation, suchas D235Y/G233V/M239V.

In a second aspect, the present invention is summarized as a kit formeasuring VWF that includes recombinant platelet GPIbα having at leasttwo mutations selected from G233V, D235Y and M239V relative to SEQ IDNO:2 or a functional fragment thereof. The kit also includes a reagentto detect a complex of VWF and GPIbα.

In some embodiments of the second aspect, the reagent can be a labeledanti-VWF antibody or labeled functional fragment thereof, such as afluorescently labeled antibody or fluorescently labeled functionalfragment thereof.

In some embodiments of the second aspect, the kit further includes anegative or positive control or both. If included, the negative controlcan be VWF-depleted plasma. If included, the positive control can bepooled plasma from individuals that do not have VWD or can be acommercially available standard, such as those available from WHO andISTH. In other embodiments of the second aspect, the kit furtherincludes an abnormal control. If included, the abnormal control can bepooled plasma from individuals with variant forms of VWD, such astype-2A, 2B or 2M VWD, as well as pooled plasma from individuals withtrue loss of in vivo VWF function or pooled plasma individuals that arenot appropriately assayed using VWF:RCo (i.e., plasma from individualshaving any gain-of-function mutation in VWF).

In some embodiments of the second aspect, the at least two mutations canbe selected from D235Y/G233V, D235Y/M239V or G233V/M239V. In otherembodiments of the second aspect, the at least two mutations can be atriple mutation, such as D235Y/G233V/M239V.

In some embodiments of the second aspect, the recombinant platelet GPIbαhaving at least two mutations selected from G233V, D235Y and M239Vrelative to SEQ ID NO:2 or functional fragment thereof can beimmobilized to a surface. In certain embodiments, the surface can be ahost cell surface of a host cell that does not natively express plateletGPIbα, as described above. In certain other embodiments, the surface canbe a solid-phase surface, as described above.

These and other features, objects and advantages of the presentinvention will become better understood from the description thatfollows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the invention. Thedescription of preferred embodiments is not intended to limit theinvention to cover all modifications, equivalents and alternatives.Reference should therefore be made to the claims recited herein forinterpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood and features, aspectsand advantages other than those set forth above will become apparentwhen consideration is given to the following detailed descriptionthereof. Such detailed description makes reference to the followingdrawings, wherein:

FIG. 1 is a schematic illustration of the platelet adhesion receptor,which shows the components of the receptor, including GPIbα, GPIbβ, GP-Vand GP-IX;

FIG. 2A shows the effect GPIbα mutations (single, double or triple).Y-axis is mean fluorescence and x-axis is log ristocetin concentrationin mg/ml), ristocetin.

FIG. 2B shows the effect GPIbα mutations (single, double or triple).Y-axis is mean fluorescence and x-axis is log ristocetin concentrationin mg/ml) and botrocetin.

FIG. 2C shows the effect GPIbα mutations (single, double or triple).Y-axis is mean fluorescence and x-axis is log botrocetin concentrationin mg/ml) during FACS. Mock is a HEK-293T cells transfected with anempty expression vector.

FIG. 3 shows an FACS assay with GPIbα having two platelet-type, pseudoVWD mutations using samples from control individuals and with type 3VWD, which has low to undetectable VWF (y-axis is mean fluorescence andx-axis is platelet poor plasma (PPP) dilutions).

FIG. 4 shows an FACS assay with additional samples from individualshaving type 2B VWD, which has gain-of-function VWF mutations (y-axis ismean fluorescence and x-axis is platelet poor plasma (PPP) dilutions).

FIG. 5 shows a FACS assay with additional samples from individualshaving type 2M VWD, which has low GPIb binding, and apparent type 2MVWD, which has low VWF:RCo/VWF:Ag, yet normal levels of VWF (y-axis ismean fluorescence and x-axis is platelet poor plasma (PPP) dilutions).

FIG. 6 shows the effect of charge on the solid-phase surface during anELISA with immobilized GPIbα having two platelet-type, pseudo VWDmutations (y-axis is mean fluorescence and x-axis is ISTH (a standard)concentration in U/dL).

While the present invention is susceptible to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail. It should beunderstood, however, that the description of preferred embodiments isnot intended to limit the invention to the particular forms disclosed,but on the contrary, the intention is to cover all modifications,equivalents and alternatives falling within the spirit and scope of theinvention as defined by the appended claims.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention stems from the inventor's observation that someindividuals with VWD have VWF mutations that lower VWF:RCo (i.e., <10IU/dL), even though their in vivo VWF function is normal (i.e., VWFstill binds to the platelet adhesion receptor component GPIb). See,Friedman K, et al., “Factitious diagnosis of type 2M von Willebranddisease (VWD) with a mutation in von Willebrand factor (VWF) thataffects the ristocetin cofactor assay but does not significantly affectVWF function in vitro,” Blood 98:536a (2001).

In contrast, other individuals with VWD (i.e., type 2B and platelet-typeVWD) have VWF or GPIbα mutations that lower the concentration ofristocetin required for platelet aggregation in an assay for VWFfunction. This paradox results from gain-of-function mutations thatcause VWF multimers and the GPIb receptors on platelets to bind moretightly to one another. The inventor hypothesized that recombinantgain-of-function GPIbα mutations could be useful in assays for VWFfunction, thereby avoiding ristocetin (i.e., ristocetinless). As usedherein, “ristocetinless” or “agonistless” means that ristocetin or otherplatelet agglutination agonists (i.e., botrocetin) are not required in aVWF assay.

The present invention therefore broadly relates to novel methods andkits for VWF utilizing gain-of-function GPIbα mutations, especiallyGPIbα mutations identified in individuals having platelet-type, pseudoVWD, to measure VWF (herein called “VWF:IbCo”). The methods and kits areuseful in a variety of applications. For example, the methods and kitsdisclosed herein may be used for diagnosing VWD in an individualsuspected of having VWD, classifying VWD in an individual diagnosed withVWD and monitoring treatment in an individual having VWD.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar to or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and materials are described herein.

As used herein, “about” means within 5% of a stated concentration range,purity range, temperature range or stated time frame.

As used herein, a “coding sequence” means a sequence that encodes aparticular polypeptide, such as GPIbα, and is a nucleic acid sequencethat is transcribed (in the case of DNA) and translated (in the case ofmRNA) into that polypeptide in vitro or in vivo when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at a 5′ (amino) terminusand a translation stop codon at a 3′ (carboxy) terminus. A codingsequence can include, but is not limited to, viral nucleic acidsequences, cDNA from prokaryotic or eukaryotic mRNA, genomic DNAsequences from prokaryotic or eukaryotic DNA, and even synthetic DNAsequences. A transcription termination sequence will usually be located3′ to the coding sequence.

As used herein, an “expression sequence” means a control sequenceoperably linked to a coding sequence.

As used herein, “control sequences” means promoter sequences,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, origins of replication, internal ribosome entrysites (“IRES”), enhancers, and the like, which collectively provide forreplication, transcription and translation of a coding sequence in arecipient cell. Not all of these control sequences need always bepresent so long as the selected coding sequence is capable of beingreplicated, transcribed and translated in an appropriate host cell.

As used herein, a “promoter” means a nucleotide region comprising anucleic acid (i.e., DNA) regulatory sequence, wherein the regulatorysequence is derived from a gene that is capable of binding RNApolymerase and initiating transcription of a downstream (3′-direction)coding sequence. Transcription promoters can include “induciblepromoters” (where expression of a polynucleotide sequence operablylinked to the promoter is induced by an analyte, cofactor, regulatoryprotein, etc.), “repressible promoters” (where expression of apolynucleotide sequence operably linked to the promoter is repressed byan analyte, cofactor, regulatory protein, etc.) and “constitutivepromoters” (where expression of a polynucleotide sequence operablylinked to the promoter is unregulated and therefore continuous).

As used herein, a “nucleic acid” sequence means a DNA or RNA sequence.The term encompasses sequences that include, but are not limited to, anyof the known base analogues of DNA and RNA such as 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5 -methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, -uracil-5-oxyacetic acid methylester, uracil-5-oxyaceticacid, pseudouracil, queosine, 2-thiocytosine and 2,6-diaminopurine.

As used herein, “operably linked” means that elements of an expressionsequence are configured so as to perform their usual function. Thus,control sequences (i.e., promoters) operably linked to a coding sequenceare capable of effecting expression of the coding sequence. The controlsequences need not be contiguous with the coding sequence, so long asthey function to direct the expression thereof. For example, interveninguntranslated, yet transcribed, sequences can be present between apromoter and a coding sequence, and the promoter sequence can still beconsidered “operably linked” to the coding sequence.

As used herein, “operable interaction” means that subunits of apolypeptide (e.g., the components of the platelet adhesion receptor,such as GPIbβ and/or GP-IX), and any other accessory proteins, that areheterologously expressed in a host cell assemble into a functioningplatelet adhesion receptor (i.e., capable of binding with VWF orfunctional fragments thereof capable of binding VWF).

As used herein, a “vector” means a replicon, such as a plasmid, phage orcosmid, to which another nucleic acid segment may be attached so as tobring about the replication of the attached segment. A vector is capableof transferring gene sequences to target cells (e.g., bacterial plasmidvectors, particulate carriers and liposomes).

Typically, the terms “vector construct,” “expression vector,” “geneexpression vector,” “gene delivery vector,” “gene transfer vector” and“expression cassette” all refer to an assembly that is capable ofdirecting the expression of a coding sequence or gene of interest. Thus,the terms include cloning and expression vehicles.

As used herein, an “isolated polynucleotide” or “isolated polypeptide”means a polynucleotide or polypeptide isolated from its naturalenvironment or prepared using synthetic methods such as those known toone of ordinary skill in the art. Complete purification is not requiredin either case. The polynucleotides and polypeptides described hereincan be isolated and purified from normally associated material inconventional ways, such that in the purified preparation thepolynucleotide or polypeptide is the predominant species in thepreparation. At the very least, the degree of purification is such thatextraneous material in the preparation does not interfere with use ofthe polynucleotide or polypeptide in the manner disclosed herein. Thepolynucleotide or polypeptide is at least about 85% pure; alternatively,at least about 95% pure; and alternatively, at least about 99% pure.

Further, an isolated polynucleotide has a structure that is notidentical to that of any naturally occurring nucleic acid molecule or tothat of any fragment of a naturally occurring genomic nucleic acidspanning more than one gene. An isolated polynucleotide also includes,without limitation, (a) a nucleic acid having a sequence of a naturallyoccurring genomic or extrachromosomal nucleic acid molecule, but whichis not flanked by the coding sequences that flank the sequence in itsnatural position; (b) a nucleic acid incorporated into a vector or intoa prokaryote or eukaryote host cell's genome such that the resultingpolynucleotide is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR) or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybrid gene (i.e., a gene encoding a fusion protein). Specificallyexcluded from this definition are nucleic acids present in mixtures ofclones, e.g., as these occur in a DNA library such as a cDNA or genomicDNA library. An isolated polynucleotide can be modified or unmodifiedDNA or RNA, whether fully or partially single-stranded ordouble-stranded or even triple-stranded. In addition, an isolatedpolynucleotide can be chemically or enzymatically modified and caninclude so-called non-standard bases such as inosine.

As used herein, “homologous” means those polynucleotides or polypeptidessharing at least about 90% or at least about 95% sequence identity to,e.g., SEQ ID NOS:1-6, respectively, that result in functionalpolypeptides that bind VWF. For example, a polypeptide that is at leastabout 90% or at least about 95% identical to the GPIbα mutationsdiscussed herein is expected to be a constituent of the plateletadhesion receptor. One of ordinary skill in the art understands thatmodifications to either the polynucleotide or the polypeptide includessubstitutions, insertions (e.g., adding no more than about tennucleotides or amino acids) and deletions (e.g., deleting no more thanabout ten nucleotides or amino acids). These modifications can beintroduced into the polynucleotide or polypeptide described belowwithout abolishing structure and ultimately, function. Polynucleotidesand/or polypeptides containing such modifications can be used in themethods of the present invention. Such polypeptides can be identified byusing the screening methods described below.

An isolated nucleic acid containing a polynucleotide (or its complement)that can hybridize to any of the uninterrupted nucleic acid sequencesdescribed above, under either stringent or moderately stringenthybridization conditions, is also within the scope of the presentinvention. Stringent hybridization conditions are defined as hybridizingat 68° C. in 5×SSC/5× Denhardt's solution/1.0% SDS, and washing in0.2×SSC/0.1% SDS+/−100 μg/ml denatured salmon sperm DNA at roomtemperature, and moderately stringent hybridization conditions aredefined as washing in the same buffer at 42° C. Additional guidanceregarding such conditions is readily available in the art, e.g., inSambrook J, et al. (eds.), “Molecular cloning: a laboratory manual,”(3rd ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001); andAusubel F, et al. (eds.), “Current Protocols in Molecular Biology,”(John Wiley & Sons, N.Y. 1995), each of which is incorporated herein byreference as if set forth in its entirety.

It is well known in the art that amino acids within the sameconservative group can typically substitute for one another withoutsubstantially affecting the function of a protein. For the purpose ofthe present invention, such conservative groups are set forth in Table 1and are based on shared properties.

TABLE 1 Amino Acid Conservative Substitutions. Original ResidueConservative Substitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, AsnAsn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E)Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe Leu (L)Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe(F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr,Phe Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

As used herein, an “antibody” means a monoclonal and polyclonal antibodyand can belong to any antibody class (i.e., IgG, IgM, IgA, etc.). One ofordinary skill in the art is familiar with methods for making monoclonalantibodies (Mab). For example, one of ordinary skill in the art can makemonoclonal antibodies by isolating lymphocytes and fusing them withmyeloma cells, thereby producing hybridomas. See, e.g., Milstein C,“Handbook of experimental immunology,” (Blackwell Scientific Pub.,1986); and Goding J, “Monoclonal antibodies: principles and practice,”(Academic Press, 1983), each of which is incorporated herein byreference as if set forth in its entirety. The cloned hybridomas arethen screened for production of, e.g., “anti-GPIbα” (i.e., antibodiesthat bind preferentially to GPIbα or fragments thereof) or “anti-VWF”antibodies (i.e., antibodies that bind preferentially to VWF orfragments thereof). Monoclonal antibodies are thus not limited by themanner in which the antibodies are produced, whether such production isin situ or not. Alternatively, antibodies can be produced by recombinantDNA technology including, but not limited, to expression in bacteria,yeast, insect cell lines or mammalian cell lines.

Likewise, one of ordinary skill in the art is familiar with methods ofmaking polyclonal antibodies. For example, one of ordinary skill in theart can make polyclonal antibodies by immunizing a suitable host animal,e.g., such as a rabbit, with an immunogen and using properly dilutedserum or isolating immunoglobulins from the serum. The animal maytherefore be inoculated with the immunogen, with blood subsequentlybeing removed from the animal and an IgG fraction purified. Othersuitable host animals include a chicken, goat, sheep, guinea pig, rat ormouse. If desired, the immunogen may be administered as a conjugate inwhich the immunogen is coupled, e.g., via a side chain of one of itsamino acid residues, to a suitable carrier. The carrier molecule istypically a physiologically acceptable carrier. The antibody obtainedmay be purified to a purity of up to about 70%, up to about 80%, up toabout 90%, up to about 95%, up to about 99% or up to about 100%.

Antibody also encompasses functional fragments, like Fab and F(ab′) 2,of anti-GPIbα or anti-VWF antibodies. Treatment of antibodies withproteolytic enzymes, such as papain and pepsin, generates these antibodyfragments, especially anti-GPIbα fragments.

Antibodies are typically conjugated to a detectable label for easyvisualization. Examples of suitable labels for the methods and kitsdescribed herein include, but are not limited to, radiolabels, biotin(which may be detected by avidin or streptavidin conjugated toperoxidase), lanthanides, alkaline phosphatase and fluorescent labels(e.g., fluorescein, rhodamine, especially the Alexa Fluor® family offluorescent dyes available from Invitrogen/Molecular Probes). Labellingof the antibody can be carried out by, e.g. labeling free amine groups(covalently or non-covalently). Some labels can be detected by using alabeled counter suitable for the detection of the label in question.

Commercially available anti-GPIbα antibodies and anti-VWF antibodies aresuitable for use with the methods and kits described herein, and can beobtained from Blood Research Institute (Milwaukee, Wis.) and Dako(Carpinteria, Calif.), respectively.

As shown in FIG. 1, the platelet adhesion receptor is comprised of acombination of four proteins, including GPIb, which is a heterodimer ofan alpha chain (GPIbα; GenBank Accession No. NM_(—)000173.4; SEQ IDNOS:1-2) and a beta chain (GPIbβ; GenBank Accession No. NM_(—)000407.4;SEQ ID NOS:3-4) linked by disulfide bonds. Other components of thereceptor include GP-V (GenBank Accession No. NM_(—)004488.2; SEQ IDNOS:5-6) and GP-IX (GenBank Accession No. NM_(—)000174.2; SEQ IDNOS:7-8). The platelet adhesion receptor binds to VWF (GenBank AccessionNo. NM_(—)000552.3; SEQ ID NOS:9-10) to regulate hemostasis andthrombosis.

Of particular interest herein is human GPIbα modified so that a plateletaggregation agonist is not required in assays of VWF function. Forexample, GPIbα can be modified to include the gain-of-function mutationsthat cause platelet-type, pseudo VWD including, but not limited to,G233V (see, Miller J, et al., “Mutation in the gene encoding the alphachain of platelet glycoprotein Ib in platelet-type von Willebranddisease,” Proc. Natl. Acad. Sci. USA 88:4761-4765 (1991), incorporatedherein by reference as if set forth in its entirety); D235V (see, DongJ, et al., “Novel gain-of-function mutations of platelet glycoproteinIBα by valine mutagenesis in the Cys209-Cys248 disulfide loop, whichinteracts with the A1 domain of VWF. Functional analysis under staticand dynamic conditions,” J. Biol. Chem. 275:27663-27670 (2000),incorporated herein by reference as if set forth in its entirety); M239V(see, Russell S & Roth G, “Pseudo-von Willebrand disease: a mutation inthe platelet glycoprotein Ib alpha gene associated with a hyperactivesurface receptor,” Blood 81:1787-1791(1993), incorporated herein byreference as if set forth in its entirety); G233S (Matsubara Y, et al.,“Identification of a novel point mutation in platelet glycoprotein Ib,Gly to Ser at residue 233, in a Japanese family with platelet-type vonWillebrand disease,” J. Thromb. Haemost. 1:2198-2205 (2003)); and K237V(see, Dong et al., supra). Advantageously, the mutation(s) can be in theCys²⁰⁹-Cys²⁴⁸ disulfide loop of GPIbα that compromise hemostasis byincreasing the affinity of GPIb for VWF. For example, and as shownbelow, the inventor found that D235Y is another gain-of-functionmutation suitable for use with the methods and kits described herein.

As used herein, a “functional fragment” means a fragment of a componentof a platelet adhesion receptor, such as a fragment of GPIbα, having atleast two of the previously mentioned mutation, yet retaining itsability to interact with VWF or other substrates. For example, the aminoterminus of GPIbα retains its ability to interact with VWF. As shownbelow, fragments of GPIbα as small as 290 amino acids and having twomutations retained an ability to interact with VWF, although smallerfragments are contemplated. With respect to VWF, a functional fragmentmay comprise at least the Al domain, which is the GPIb binding domain.With respect to antibodies, functional fragments are those portions ofan antibody that bind to a particular epitope, such as the domainsindicated above.

As used herein, a “sample” means a biological sample, such as amnioticfluid, aqueous humor, cerebrospinal fluid, interstitial fluid, lymph,plasma, pleural fluid, saliva, serum, sputum, synovial fluid, sweat,tears, urine, breast milk or tissue that has or is suspected of havingVWF. With respect to measuring VWF, plasma is a suitable sample.

As used herein, a “surface” means, e.g., a cell surface or solid-phasesurface, such as an unsoluble polymer material, which can be an organicpolymer, such as polyamide or a vinyl polymer (e.g., poly(meth)acrylate,polystyrene and polyvinyl alcohol or derivates thereof), a naturalpolymer, such as cellulose, dextrane, agarose, chitin and polyaminoacids, or an inorganic polymer, such as glass or plastic. Thesolid-phase surface can be in the form of a bead, microcarrier,particle, membrane, strip, paper, film, pearl or plate, particularly amicrotiter plate.

One aspect of the present invention includes a diagnostic assay formeasuring VWF. The underlying methodology of the assay can be FC, FACSor ELISA, each of which is well known to one of ordinary skill in theart. See, e.g., Alice Giva, “Flow cytometry: first principles,” (2nd ed.Wiley-Liss, New York, 2001); Howard Shapiro, “Practical flow cytometry,”(4th Ed. Wiley-Liss, New York, 2003); Larry Sklar, “Flow cytometry forbiotechnology,” (Oxford University Press, New York, 2005); J. PaulRobinson, et al., “Handbook of flow cytometry,” (Wiley-Liss, New York,1993); “Flow cytometry in clinical diagnosis,” (3rd ed., Carey, McCoyand Keren, eds., ASCP Press 2001); Lequin R, “Enzyme immunoassay(EIA)/enzyme-linked immunosorbent assay (ELISA),” Clin. Chem.51:2415-2418 (2005); Wide L & Porath J, “Radioimmunoassay of proteinswith the use of Sephadex-coupled antibodies,” Biochem. Biophys. Acta.30:257-260 (1966); Engvall E & Perlman P, “Enzyme-linked immunosorbentassay (ELISA). Quantitative assay of immunoglobulin G,” Immunochemistry8:871-874 (1971); and Van Weemen B & Schuurs A, “Immunoassay usingantigen-enzyme conjugates,” FEBS Letters 15:232-236 (1971), each ofwhich is incorporated herein by reference as if set forth in itsentirety.

As noted above, the surface for the methods and kits described hereincan be a host cell surface expressing at least platelet GPIbα for use inFACS. For example, one can heterologously express (either transiently orstably) mutant GPIbα or other components of platelet adhesion receptor(i.e., GPIbβ and/or GP-IX) in host cells. Methods of expressingpolynucleotides and their encoded platelet glycoprotein receptorpolypeptides in heterologous host cells are known to one of ordinaryskill in the art. See, e.g., Tait A, et al., “Phenotype changesresulting in high-affinity binding of von Willebrand factor torecombinant glycoprotein Ib-IX: analysis of the platelet-type vonWillebrand disease mutations,” Blood 98:1812-1818 (2001), incorporatedherein by reference as if set forth in its entirety; and Dong et al.,supra.

Cells suitable for use herein preferably do not natively display GPIbαor the other components of the platelet adhesion receptor complex. Onesuch cell type is HEK-293T cells (American Type Culture Collection(ATCC); Manassas, Va.; Catalog No. CRL-11268). See also, Graham F, etal., “Characteristics of a human cell line transformed by DNA from humanadenovirus type 5,” J. Gen. Virol. 36:59-74 (1977), incorporated hereinby reference as if set forth in its entirety. HEK-293 cells are easy toreproduce and to maintain, are amenable to transfection using a widevariety of methods, have a high efficiency of transfection and proteinproduction, have faithful translation and processing of proteins andhave a small cell size with minimal processes appropriate forelectrophysiological experiments.

Another suitable cell type is COS-7 cells (ATCC; Catalog No. CRL-1651).See also, Gluzman Y, “SV40-transformed simian cells support thereplication of early SV40 mutants,” Cell 23:175-182 (1981), incorporatedherein by reference as if set forth in its entirety. Like HEK-293 cells,COS-7 cells are easy to reproduce and maintain and are amenable totransfection using a wide variety of methods.

Yet another suitable cell type is Xenopus oocytes. Xenopus oocytes arecommonly used for heterologous gene expression because of their largesize (˜1.0 mm), which makes their handling and manipulation easy.Xenopus oocytes are readily amenable to injection, and thus expressfunctional proteins when injected with cRNA for an desired protein.

Yet another suitable cell type is S2 Drosophila melanogaster cells. S2cells are ideal for difficult-to-express proteins, and a S2 expressionsystem is commercially available from Invitrogen (Carlsbad, Calif.). TheS2 expression system can be engineered to preferably lack the Bipsecretion sequence so that the encoded proteins are expressed on thecell surface. Expression of platelet adhesion receptor components in S2cells was previously shown by Celikel et al. See, Celikel R, et al.,“Modulation of alpha-thrombin function by distinct interactions withplatelet glycoprotein Ibα,” Science 301:218-221 (2003), incorporatedherein by reference as if set forth in its entirety.

Any of the contemplated polynucleotides for the platelet adhesionreceptor can be cloned into an expression vector (or plurality ofexpression vectors) engineered to support expression from thepolynucleotides. Suitable expression vectors comprise a transcriptionalpromoter active in a recipient host cell upstream of, e.g., a GPIbαpolynucleotide engineered to have the previously mentioned mutations oradditional polynucleotides and can optionally comprise a polyA-additionsequence downstream of the polynucleotide.

The vector(s) can be introduced (or co-introduced) by, for example,transfection or lipofection, into recipient host cells competent toreceive and express mutant GPIbα and optionally other components of theplatelet adhesion receptor. A commercially available lipofection kit,such as a kit available from Mims Corporation (Madison, Wis.) can beemployed. Preferably, the recipient host cells do not natively containGPIbα, so that the presence of it is completely attributable toexpression from the introduced expression vector. Suitable recipienthost cells are described above and can express the polypeptides on theirsurface or secrete them.

Alternatively, the surface for the methods and kits described herein canbe a solid-phase surface having platelet GPIbα immobilized thereupon by,e.g., covalent attachment or antibodies. Suitable solid-phase surfacesinclude the solid-phase surfaces described above. One of ordinary skillin the art is familiar with methods for attaching anti-GPIbα antibodiesor functional fragments thereof to solid-phase surfaces. For example,the antibody or functional fragment thereof can be immobilized on thesurface directly by covalent coupling or via a carrier such as a linkermolecule or an antibody immobilized on the solid-phase surface. Theantibody can be a polyclonal or monoclonal antibody, such as anti-GPIbαor a functional fragment thereof. Alternatively, the antibody can be ananti-epitope antibody that recognizes an epitope-tag (e.g., biotin,digoxigenin, GST, hexahistidine, hemagglutinin, FLAG™, c-Myc, VSV-G, V5and HSV) complexed with GPIbα. Commercially available epitope tags andtheir respective antibodies are suitable for use with the methods andkits described herein, and can be obtained from Sigma Aldrich (St.Louis, Mo.) and Abcam, Inc. (Cambridge, Mass.).

The methods and kits described herein are thus sensitive to themeasurement of the more functional, large VWF multimers, correlates withVWF:Ag in individuals with reduced VWF function, and remains unaffectedby mutations that affect VWF binding of ristocetin but do not have ableeding phenotype.

The invention will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Example 1 Cells Heterologously Expressing Mutant GPIbαSpontaneous Binding in the Absence Ristocetin

Methods: A heterologous platelet adhesion receptor expression system wasconstructed by transiently transfecting HEK-293T cells (ATCC) with afull-length GPIbα construct encoding a single mutation (i.e., G233V,D235Y or M239V), a double mutation (i.e., G233V/M239V, G233V/D235Y orD235Y/M239V) a triple mutation (i.e., G233V/D235Y/M239V) relative to SEQID NO:2 or wild-type GPIbα (SEQ ID NO:2). Some HEK-293T cells also weretransiently transfected with GPIbβ and GP-IX constructs encoding SEQ IDNOS:4 and 8, respectively. A mock group of HEK-293T cells were treatedsimilarly, but were transfected with an expression vector lacking theabove constructs, thereby serving as controls.

The constructs were cloned in to a pCI-neo vector (Promega; Madison,Wis.) and expressed in HEK-293T cells as described below. In someinstances, separate constructs were made for each GPIbα mutation;however, in other instances, a single construct was made having multipleGPIbα mutations.

Briefly, HEK-293T cells were first seeded until they were 50-80%confluent (i.e., 3.5-4×10⁶/100 mm dish). Typically, the cells wereseeded the day before transfection. For transfection, Hanks BalancedSalt Solution (HBSS) and OptiMEM (Invitrogen) were warmed to 37° C. 800μl of OptiMEM was added to 17×100 polystyrene tubes (2 tubes/plate to betransfected). The following was added to one set of tubes: 4.5 μg of DNA(1.5 μg of each construct) and 20 μl PLUS Reagent (Invitrogen). Thefollowing was added to another set of tubes: 30 μl Lipofectamine(Invitrogen). Each set was allowed to incubate at room temperature for15 minutes. The DNA mixture was then added to the Lipofectamine mixtureand incubated at room temperature for 15 minutes. During incubation, thecells were washed twice with 5 ml HBSS. 3.4 ml of OptiMEM was added tothe DNA/Lipofectamine mixture, and then added to the HEK-293T cells(total volume=5 ml). The cells were then incubated at 37° C. with 5% CO₂for 3-3.5 hours.

Following transfection, the transfection medium was removed and 8 ml offresh complete medium was added to the cells. The cells were thenincubated at 37° C. with 5% CO₂ for about 60 hours. Cells were thenharvested for use in a standard FACS assay using ristocetin.

For FACS, about 50 μl of a 1:10 dilution of platelet poor plasma (PPP;source of VWF) in assay buffer was added to the plate and seriallydiluted 1:2 to final dilution of 1:80. ISTH Lot #3 (GTI; Milwaukee,Wis.) was used as a standard and diluted 1:10 in assay buffer andserially diluted 1:2 to a final dilution of 1:320. The plate was thenincubated for one hour at room temperature. After the one-hourincubation, the plate was centrifuged again at 1200 rpm for 5 minutesand the supernatant was discarded.

In some experiments, the PPP was diluted in PBS containing 1% BSA andeither 1 mg/ml Ristocetin A (American Biochemical & Pharmaceuticals,Ltd.; Marlton, N.J.) or 1 mg/ml Botrocetin (Sigma Aldrich).

Fluorescently labeled antibodies (anti-GPIbα; Blood Research Institute)were diluted to a final concentration of 5 μg/ml in assay buffer.Fluorescently labeled anti-VWF polyclonal was also was diluted to afinal concentration of 5 μg/ml in assay buffer and added to transfectedcells incubated in PPP. Normal rabbit IgG (NRIgG; Pierce) and AP-1 wereadded at a concentration of 5 μg/ml to transfected cells as negative andpositive controls, respectively. The plate was then incubated in thedark for one hour at room temperature. Assay buffer was added to eachwell to bring the final volume to 150 μl, and FACS was performed using aBD LSRII System (Becton Dickinson). Results are shown in VWF:IbCo units.

Results: As shown in FIG. 2A, mock transfected HEK-293T cells did notshow any binding in the presence of ristocetin, while cells expressingwild-type GPIbα showed a concentration-dependent decrease in ristocetinbinding after 1.2 mg/ml. HEK-293T cells expressing only one of the GPIbαmutations showed increased sensitivity even at low concentrations ofristocetin, which suggests that the binding is independent ofristocetin. Cells expressing two GPIbα mutations showed an extremesensitivity to ristocetin or alternatively, an increased spontaneousbinding that was independent of ristocetin. HEK-293T cells expressingthe triple GPIbα mutation (i.e., G233V/D235Y/M239V), however, did notshow increased sensitivity/spontaneous binding relative to the doublemutants. As shown in FIG. 2B, each of the double mutants (i.e.,G233V/M239V, G233V/D235Y or D235Y/M239V) showed comparable spontaneousbinding relative to one another that was not significantly affected byristocetin (i.e., ristocetinless). As expected the, wild-type controlshowed concentration-dependent increases in VWF:IbCo to ristocetin. Asshown in FIG. 2C, VWF:IbCo is not affected by the type of plateletaggregation agonist, as none of the double mutants was significantlyaffected by botrocetin (i.e., botrocetinless). As expected, wild-typecontrol showed concentration-dependent increases in VWF:IbCo tobotrocetin.

Example 2 VWF Function in Patient Samples Using Mutant GPIbα in FACS

Methods: HEK293T cells were transiently transfected with a wild-typeGPIbα construct or GPIbα encoding one of the double mutants, asdescribed above. The cells were additionally transfected with the GPIbβand GP-IX constructs. A group of HEK-293T cells were mock transfected,as describe above.

After forty-eight hours, the transfected cells were lifted from theplate with 3 mM EDTA, resuspended in assay buffer (i.e., 1× PBScontaining 2% BSA) and counted. Trypsin was not used, as it potentiallycan cleave GPIbα from the cell surface. After counting, 1.75×10⁵ cellswere plated 96-well plate (Becton Dickinson; Franklin Lakes, N.J.) as away of standardizing GPIbα on the plate surface, and the plate was thencentrifuged at 2000 rpm for 5 minutes to pellet the cells. Thesupernatant was discarded.

HEK-293T cells expressing the GPIbα mutations were used in flowcytometry assays to test VWF binding, which was measured with afluorescently labeled anti-VWF polyclonal antibody from Dako. A normalcurve was developed using serial dilutions of reference plasmapreviously standardized against both the ISTH and WHO VWF standardsbased on the VWF:Ag international standard that is also standardized forVWF:RCo.

In one set of experiments, normal patient samples were used to determinewhether the HEK-293T cells required all components of the plateletadhesion receptor or simply GPIbα. Normal patient samples were used. Inanother set of experiments, patient samples from normal individuals andindividuals having VWD were used in the FACS assay as described above inExample 2.

Samples included 41 normals, 16 type-2M VWD, 5 type-2B VWD and 5 type-2AVWD plasma, Included therein were individuals with apparent type-2M VWD,but without clinical symptoms, and African Americans with a reducedVWF:RCo/VWF:Ag (RCo/Ag) ratio. Of the 16 type-2M VWD samples, 7 hadmarkedly reduced VWF:IbCo (consistent with the VWF:RCo assay), and 9 hadnormal VWF:IbCo. African Americans with SNPs associated with reducedRCo/Ag ratios had VWF:IbCo assays that correlated with their VWF:Ag incontrast to the abnormal RCo/Ag ratios identified by standard assays.Type-2A individuals exhibited reduced VWF:IbCo assays and multimer sizeseemed to correlate with VWF:IbCo activity. Thus, measurement of VWFfunction using the VWF:IbCo assay more directly correlates with VWFfunction and avoids some of the pitfalls and functional variability ofVWF:RCo assays.

Results: As shown in Table 2, GPIbβ and GP-IX are not required forsurface expression of the mutant GPIbα, as FACS results from HEK-293Tcells expressing multiple components of the plate adhesion receptor werenot significantly different from cells expressing only GPIbα.

TABLE 2 Effect of GPIbα Having a Double Mutation With or Without theOther Platelet Adhesion Receptor Components in a FACS. % Diff. % GPIbα %Diff btw Diff. (G233V/ btw GPIbα, btw Known M239V), Trans- GPIbβ GPIbαVWF:RCo GPIbβ fec- and IX & Sample (IU/dL) and IX GPIbα tions & KnownKnown ISTH 2 71 70.4 70.3 0.1 0.4 0.5 ISTH 3 86 86.9 91.4 2.5 0.5 3.0CCNRP 82 to 103 89.0 74.6 8.8 4.1 4.7 Cntrl 3 65 64.4 52.1 10.5 0.5 11.0Cntrl 4 24.6 26.7 22.7 8.0 4.0 4.1 JS 0 0 0 0 0 0 XX-01 200 136.4 119.56.6 18.9 25.2 ISTH = reference sample

As shown below in Table 3, the FACS assay resulted in VWF measurementscomparable to a method used in clinical laboratories. Samples werenormal individuals and individuals having VWD. Table 4 is similar toTable 3, except that the samples were from normal individuals andindividuals having Type 2 VWD. Table 5 is also similar to Table 3,except that the samples were from individuals having Type 2M VWD.

TABLE 3 Summary of VWF:IbCo by FACS in Plasma Samples from AfricanAmericans and Caucasians with and without VWF Single NucleotidePolymorphisms (SNPs).

1 = DT method (a clinical laboratory method) 2 = BRI method (BloodResearch Institute method) Shaded area = <0.81

TABLE 4 VWF:IbCo by FACS in Plasma from African Americans and CaucasiansWith/Without Type 2 VWD and Repeats. VWD VWF Sample Race PhenotypeMutation VWF:Ag VWF:RCo RCo/Ag FACS1 FACS1/Ag FACS2 FACS2/Ag DB AA ″2M″3 AA snps 86 47 0.547 78 0.910 78 0.905 MK0055 AA ″2M″ P1467S 257  360.140 214 0.833 184 0.718 LJ C ″2M″ 3 AA snps 66 40 0.606 180 2.734 480.735 IN0061 2M R1374C 22 11 0.500 4 0.204 10 0.432 RH 2B R1308S 43 370.860 67 1.558 67 1.557 LB 2B V1316M 91 62 0.681 159 1.751 106 1.162 SB2B V1316M 27 12 0.444 36 1.347 25 0.914 AJ 2B H1268D 21 17 0.810 311.484 41 1.959 PB0068 2B R1306W 23 13 0.565 — — 25 1.065 YG 2A L1503P 2613 0.500 — — 19 0.714 AV 2A G1579R 46 16 0.348 — — 1 0.028 AT0021 2AM7401? 31 12 0.387 — — 18 0.574 AT0032 2A I1628T 120  32 0.267 — — 1030.586 IA0001 2A R1597W 33 <10 — — — 8 0.247 AT0017 AA NL 3 AA snps 225 95 0.422 144 0.641 156 0.695 XX0027 AA NL 3 AA snps 195  130 0.677 1470.753 116 0.595 XX0004 C NL — 96 109 1.135 128 1.334 114 1.183 XX0013 CNL — 124  169 1.363 186 1.503 105 0.843 PB0014 AA NL — 234  211 0.902197 0.843 213 0.909 AT0042 AA NL — 86 69 0.802 64 0.739 91 1.056 AA =African American C = Caucasian NL = normal ″2M″ = apparent type 2M

TABLE 5 VWF Function in Plasma from African Americans and CaucasiansWith/Without Type 2M VWD. VWD VWF Sample Race Phenotype Mutation VWF:Ag1 VWF:RCo 1 RCo/Ag 1 FACS2 FACS2/Ag TB C ″2M″ — 127 87 0.69 87 0.69 DBAA ″2M″ 3 AA snps 86 47 0.55 78 0.91 AC C 2M G13242S 95 13 0.14 <1.1 —BF — 2M I1416T (new) 89 31 0.35 36 0.41 MG H 2M I1425F 45 16 0.36 >1.1 —LG C 2M E1359K 67 37 0.55 27 0.41 GI — 2M D1283H (new) 16 4 0.25 <1.1 —KJ C 2M — 12 3 0.25 <1.1 — LJ AA ″2M″ 3 AA snps 66 40 0.61 180 2.73 BM C2M I1426T 156 43 0.28 93 0.60 AR — 2M R1374L 48 10 0.21 <1.1 — DR AA″2M″ R1342C; 38 12 0.32 37 0.97 I1343V; 1301- 3103 del; and R2185QMK0038 C 2M R1392-Q1402 47 11 0.23 <1.1 — del IN0061 C 2M R1374C 22 110.50 4 0.20 MK0055 AA ″2M″ P1467S 257 36 0.14 214 0.83 MK0058 AA ″2M″P1467S 265 68 0.14 194 0.73 AA = African American C = Caucasian H =Hispanic

Results: As shown in FIG. 4, individuals with normal VWF showed atypical increase in mean fluorescence with lower dilutions of theirplasma. As expected, individuals with Type 3 VWD showed change in meanfluorescence because their plasma has low or no VWF.

As shown in FIG. 5, individuals with Type 2B VWD showed a much earlierincrease in mean fluorescence when compared to normals, starting at veryhigh dilutions of their plasma (i.e., >1/100). Type 2B VWD ischaracterized as having gain-of-function mutations. Again, individualswith Type 3 VWD showed no reaction in the assay.

As shown in FIG. 6, individuals with Type 2M VWD showed no increase inmean fluorescence when compared to normals. Type 2M VWD is characterizedby defective VWF that does not interact with GPIbα. Individuals withapparent Type 2M (“2M”) showed a much earlier increase in meanfluorescence when compared to normals, starting at very high dilutionsof their plasma (i.e., >1/100). Apparent Type 2M is characterized by lowVWF:RCoNWF:Ag, yet normal levels of VWF. Again, individuals with Type 3VWD showed no reaction in the assay.

Example 3 Mutant GPIbα Function in ELISA

S2 cells (Invitrogen) were stably transfected with a mutant GPIbαconstruct, a wild-type GPIbα construct and a GP-IX construct. In someexperiments, S2 cells were transfected with GPIbα constructs having aC65A mutation and ΔTM290 mutation. The C65A mutation removed a cysteinethat could potential allow dimerization of GPIbα; and the ΔTM290mutation removed the transmembrane region so that the expressed proteinwas excreted.

Briefly, the constructs were cloned into a pMT/Bip/V5-His:GPIbαC65A,D235Y,M239V ΔTM290 or pMT/Bip/V5-His:GPIbα C65A ΔTM290 secretionvector (Invitrogen). On day 1, S2 cells were counted and seeded into a35 mm dish or a well of a 6 well plate at 3×10⁶ cells in 3 ml ofcomplete medium (Ex-Cell 420+10% FBS+7 mM L-Glutamine). The cells wereallowed to grow 6-8 hours at 28° C. The following was added to one setof tubes: Solution A, which contained 36 μl of 2M CaCl₂, 19 μg ofplasmid DNA (purified with Qiagen Maxi Kit; Qiagen; Valencia, Calif.), 1μg pCoBlast (selection vector) and ddH₂O up to 300 μl. The following wasadded to another set of tubes: Solution B, which contained 300 μl of 2×HEPES buffered saline. Solution A was slowly added dropwise to solutionB while gently vortexing. The combined solutions then were incubated atroom temperature for 30-40 minutes until a fine precipitate formed. Themixed solution was added dropwise to the plated cells while gentlyswirling the plate. The cells were then incubated overnight at 28° C.(about 16-24 hours).

The next day, the transfection solution was removed and replaced with 3ml of fresh complete medium and incubated at 28° C. without CO₂. On day5, the cells were resuspended cells and transferred to a 15 cc conicaltube, centrifuged at 2400 rpm for 2 minutes. The medium was decanted,and the cells were resuspended in 3 ml of stable medium (completemedium+25 μg/ml Blastidin-S) and plated in a new dish or well.

Selection began on week 2. As done on Day 5, the selection medium wasreplaced every 3-4 days with 3 ml fresh selection medium. Selection andexpansion continued through week 3. During this time, the cells wereresuspended, transferred to 15 cc conical tubes, and centrifuged at 2400rpm for 2 minutes. The media as decanted, and the cells were resuspendedin 5 ml of selector media and plated in new T25 flask. After 4 days, thecells were expanded from 1 T25 to 2 T25 flasks.

Expansion and freezing stocks began on week 4. Cells were expanded fromthe T25 flasks to T75 flasks (3×10⁶ cells/ml medium). T75 flasksreceived 15 ml medium, which was about 45×10⁶ cells. The remaining cells(about 2×10⁷ cells/vial) were frozen and stored in liquid nitrogen.

Induction of the cells in the T75 flasks began on week 5. Cells wereresuspended, transferred to a 15 cc conical tube for counting andcentrifuged. 45×10⁶ cells were resuspended 15 ml induction medium(stable medium+500 μM CuSO₄) and transferred to T75 flasks. The cellswere then incubated 4 days at 28° C., the supernatant having secretedGPIbα was harvested.

As shown Table 6 and FIG. 3, various solid-phase surfaces were firsttested for the ELISA assays. Table 6 shows that the surface density ofGPIbα was affected by the surface charge of the solid-phase surface;whereas FIG. 3 shows that different solid-phase surfaces coated withGPIbα having a double mutation affected VWF binding. Solid-phase surfacecharge appeared to affect GPIbα/VWF binding, suggesting that anysolid-phase surface should first be tested for it ability (1) to providea uniform density of GPIbα and (2) to permit VWF to bind to the GPIbα.After considering both Table 6, and FIG. 3, Immulon® 4 HBX Plates workedbest and were used thereafter.

TABLE 6 Effect of Various Solid-Phase Surfaces on Concentration of GPIbαDouble Mutation (G233V/M239V) (same samples on different plates).Calculated Solid-Phase Surface Characteristic of the Surfaceconcentration GPIbα Immulon 1 Hydrophobic 635.1 Immulon 2 Hydrophobic370.8 Immulon 4 Maximum 383.7 Polysorp Hydrophobic 321.7 Corning MediumHydrophobic 576.8 Corning High Ionic and/or Hydrophobic 414.7 MultisorbPolar Molecules No binding Maxisorb Hydrophobic/Hydrophilic 408.5

An Immulon 4 HBX Plate (Thermo Scientific; Waltham, Mass.) was coatedwith anti-GPIbα monoclonal antibody 142.16 (Blood Research Institute) ata concentration of 5 μg/ml, which was then incubated overnight at 4° C.The plate was blocked with PBS containing 1% BSA for 1 hour at roomtemperature. Nickel-purified S2-expressed proteins—GPIbα C65A, D235Y,M239V and ΔTM290—were diluted in PBS containing 1% BSA and incubated onthe anti-GPIbα antibody-coated plate for 1 hour at 37° C. See, Celikelet al., supra.

PPP from controls or individuals having VWD was diluted 1:50 in PBScontaining 1% BSA and serially diluted 1:2 to a final dilution of 1:100.Diluted PPP was added to the plate and incubated for 1 hour at 37° C.ISTH Lot#3 was again used as a standard, with curve dilutions startingat 1:25 in substrate buffer, which was then serially diluted 1:2 to afinal dilution of 1:1600. 2 μg/ml biotinylated AVW-1 and AVW-15 (BloodResearch Institute) were added to the plate and incubated for 30 minutesat 37° C. Finally, streptavidin-conjugated alkaline phosphatase (JacksonImmunoResearch Laboratories, Ltd.; West Grove, Pa.), diluted 1:5000 insubstrate buffer, was added to the plate and incubated for 30 minutes at37° C. p-Nitrophenyl Phosphate (PNPP; Invitrogen), an alkaline phosphatesubstrate, was diluted 1:100 in substrate buffer and added to the plate.The plate was read at 405/650 nm on a plate reader. The plate was washedthree times between each step with PBS containing 0.05% Tween-20.

Results: As shown in Tables 7 and 8, individuals with normal VWF showedsimilar ELISA results whether ristocetin was added to the assay or not.In addition, the ELISA assay resulted in VWF measurements comparable toa method used in clinical laboratories

TABLE 7 Summary of VWF:IbCo by ELISA in Plasma Samples from AfricanAmericans and Caucasians with and without VWF Single NucleotidePolymorphisms (SNPs). IbCo Ristocetin Clinical VWF:RCo/ Subject VWF:AgELISA ELISA IbCo/VWF:Ag Ris/VWF:Ag VWF:Ag VWF:RCo VWF:Ag ISTH 3 A 121.25109.4 127.3 0.90 1.05 106 86 0.81 ctrl 5 (70%) 75.62 68.97 69.68 0.910.92 74.2 60.2 0.81 ctrl 6 (35%) 32.28 31.12 28.76 0.96 0.89 37.1 30.10.81 CCNRP 7122 94.56 84.6 73.34 0.89 0.78 114 71 0.62 A ISTH 3 B 96.7199.71 104.05 1.03 1.08 106 86 0.81 MK0038 33.44 1.41 14.16 0.04 0.42 4711 0.23 XX0017 139.5 157.35 169.5 1.13 1.22 206 200 0.97 JS 0 0.5 0.990.00 0.00 <1 <10 0.00 ctrl 8 (30%) 23.84 26.96 23.58 1.13 0.99 31.8 25.80.81 ISTH 3 C 85.14 94.42 90.48 1.11 1.06 106 86 0.81 CCNRP 7122 59.6160.64 55.44 1.02 0.93 114 71 0.62 B AT0068 70.4 59.18 31.47 0.84 0.45 9957 0.58

TABLE 8 Summary of VWF:IbCo by ELISA in Plasma Samples from AfricanAmericans and Caucasians with and without VWF Single NucleotidePolymorphisms (SNPs). Clinical IbCo Clinical Clinical IbCo RistocetinVWF:RCo/V ELISA/BRI Subject VWF:Ag BRI VWF:Ag VWF:RCo ELISA ELISA WF:AgVWF:Ag AA w/1380 + 1435 + 1472 HN 334 228 165 165 — 0.494 0.725 XX 278228 225 235 222 0.809 1.029 AT 257 309 248 234 220 0.965 0.759 AT 225159 198 149 152 0.880 0.932 AT 225 172 95 67 106 0.422 0.393 IN 215 179104 77 73 0.484 0.429 XX 193 200 140 123 154 0.725 0.616 NO 179 178 180171 — 1.006 0.960 AT 103 83 69 58 64 0.670 0.701 XX 85 67 74 73 57 0.8711.095 AT 71 65 72 70 53 1.014 1.077 HN 67 77 54 54 — 0.806 0.704 AAw/1472 alone NO 259 209 224 151 213 0.865 0.723 XX 195 129 130 118 1320.667 0.910 XX 185 143 154 143 183 0.832 1.001 XX 167 172 170 123 1981.018 0.714 NO 166 151 175 155 — 1.054 1.025 IN 153 123 146 120 55 0.9540.970 NO 144 141 85 92 — 0.590 0.652 DT 141 — 121 — — 0.858 — HN 139 —98 — — 0.705 — XX 137 112 123 90 151 0.898 0.801 HN 136 136 113 106 —0.831 0.784 XX 122 89 85 75 — 0.697 0.839 XX 116 103 89 82 81 0.7670.793 XX 110 104 91 100 62 0.827 0.967 IN 108 107 101 86 94 0.935 0.800AT 99 91 57 50 25 0.576 0.550 DT 98 89 85 79 85 0.867 0.885 AT 84 96 7982 63 0.940 0.856 AA w/no SNPs NO 243 237 252 217 — 1.037 0.917 PB 234192 211 110 83 0.902 0.576 DT 224 185 167 190 — 0.746 1.025 AT 199 178193 177 — 0.970 0.993 NO 195 179 220 207 233 1.128 1.160 AT 164 132 15198 96 0.921 0.743 AT 154 139 176 159 135 1.143 1.143 IN 122 76 85 68 740.697 0.897 PB 109 91 93 76 63 0.853 0.832 PB 86 63 88 64 52 1.023 1.025AT 86 107 97 68 63 1.128 0.633 AT 86 57 69 60 56 0.802 1.055 XX 85 79 9265 44 1.082 0.817 AT 82 93 94 70 49 1.146 0.750 C w/1380 + 1435 + 1472PB 180 144 149 122 115 0.828 0.842 IN 94 91 84 68 79 0.894 0.747 Cw/1472 alone XX 206 254 200 266 251 0.971 1.050 IN 192 137 144 133 1260.750 0.973 DT 174 148 137 165 127 0.787 1.119 IN 171 106 122 94 1270.713 0.888 PB 129 102 85 76 66 0.659 0.751 IN 111 88 99 76 78 0.8920.861 XX 97 67 89 82 80 0.918 1.224 HN 94 103 82 82 — 0.872 0.791 IN 9165 88 58 51 0.967 0.902 C w/no SNPs PB 289 313 256 309 292 0.886 0.988IN 237 171 255 154 275 1.076 0.901 IN 187 165 138 124 144 0.738 0.753 XX129 121 149 90 112 1.155 0.745 XX 124 128 169 137 127 1.363 1.073 IN 10382 92 67 78 0.893 0.815 IN 100 71 91 68 72 0.910 0.957 XX 96 93 109 132103 1.135 1.425 IN 96 86 110 94 87 1.146 1.086 XX 94 77 101 74 83 1.0740.972 XX 94 100 86 88 93 0.915 0.875 DT 88 90 107 91 83 1.216 1.008 PB88 79 78 50 54 0.886 0.628 IN 85 61 77 58 57 0.906 0.961 IN 85 74 82 6556 0.965 0.872 PB 83 80 88 60 51 1.060 0.742 DT 82 73 79 65 63 0.9630.891 PB 74 52 69 53 34 0.932 1.017 DT 68 57 71 55 56 1.044 0.956 XX 5854 61 52 49 1.052 0.958 AA = African American C = Caucasian

Thus, measurement of VWF function using a VWF:IbCo FACS or ELISA assaymore directly correlates with VWF function and avoids some of thepitfalls and functional variability observed with VWF:RCo assays.

The invention has been described in connection with what are presentlyconsidered to be the most practical and preferred embodiments. However,the present invention has been presented by way of illustration and isnot intended to be limited to the disclosed embodiments. Accordingly,those skilled in the art will realize that the invention is intended toencompass all modifications and alternative arrangements within thespirit and scope of the invention as set forth in the appended claims.

The invention claimed is:
 1. A method of measuring von Willebrand factor(VWF) without using a platelet agglutination agonist, the methodcomprising the steps of: providing a surface comprising immobilizedplatelet glycoprotein Ibα (GPIbα) or a functional fragment thereof,wherein the immobilized GPIbα or functional fragment thereof comprisesat least two mutations selected from the group consisting of G233V,D235Y and M239V relative to SEQ ID NO:2; contacting a sample having orsuspected of having VWF with the surface, wherein the contacting is donewithout a platelet aggregation agonist; and detecting a complex of VWFand GPIbα.
 2. The method of claim 1, wherein the surface is a host cellsurface, and wherein the host cell does not natively express GPIbα. 3.The method of claim 2, wherein the host cell for the host cell surfaceis selected from the group consisting of a Xenopus oocyte, a CHO-K1cell, a L929 cell, a HEK-293T cell, a COS-7 cell and a S2 cell, andwherein the host cell is engineered to comprise a polynucleotideencoding GPIbα or functional fragment thereof having the at least twomutations selected from the group consisting of G233V, D235Y and M239Vrelative to SEQ ID NO:2.
 4. The method of claim 2, wherein the host cellsurface also comprises glycoprotein Ibβ (GPIbβ) and optionallyglycoprotein IX (GP-IX), wherein GPIbβ comprises SEQ ID NO:4 and GP-IXcomprises SEQ ID NO:8.
 5. The method of claim 1, wherein the surface isa solid-phase surface selected from the group consisting of agarose,glass, latex and plastic.
 6. The method of claim 5, wherein thesolid-phase surface comprises an anti-GPIbα antibody that binds theGPIbα or functional fragment thereof.
 7. The method of claim 1, whereinthe sample is plasma.
 8. The method of claim 1, wherein the at least twomutations are selected from the group consisting of D235Y/G233V,D235Y/M239V and G233V/M239V.
 9. The method of claim 1, wherein the atleast two mutations are D235Y/G233V/M239V.
 10. The method of claim 1,wherein a labeled anti-VWF antibody is used to detect the complex of VWFand GPIbα.
 11. A kit for measuring active von Willebrand factor (VWF),the kit comprising: recombinant platelet glycoprotein Ibα (GPIbα) or afunctional fragment thereof, wherein the GPIbα or functional fragmentthereof comprises at least two mutations selected from the groupconsisting of G233V, D235Y and M239V relative to SEQ ID NO:2; and areagent to detect a complex of VWF and GPIbα.
 12. The kit of claim 11,wherein the reagent is a labeled anti-VWF antibody.
 13. The kit of claim11, further comprising a control, wherein the control is a plasma samplefrom an individual that does not have von Willebrand disease (VWD). 14.The kit of claim 11, wherein the GPIbα or functional fragment thereofhaving the at least two mutations selected from the group consisting ofG233V, D235Y and M239V relative to SEQ ID NO:2 is immobilized on asurface.
 15. The kit of claim 14, wherein the surface is a host cellsurface, and wherein the host cell does not natively express GPIbα. 16.The kit of claim 15, wherein the host cell for the host cell surface isselected from the group consisting of a Xenopus oocyte, a CHO-K1 cell, aL929 cell, a HEK-293T cell, a COS-7 cell and a S2 cell, and wherein thehost cell is engineered to comprise a polynucleotide encoding GPIbα orfunctional fragment thereof having the at least two mutations selectedfrom the group consisting of G233V, D235Y and M239V relative to SEQ IDNO:2.
 17. The kit of claim 15, wherein the host cell surface alsocomprises glycoprotein 1bβ (GPIbβ) and optionally glycoprotein IX(GP-IX), wherein GPIbβ comprises SEQ ID NO:4 and GP-IX comprises SEQ IDNO:8.
 18. The kit of claim 14, wherein the surface is a solid-phasesurface selected from the group consisting of agarose, glass, latex andplastic.
 19. The kit of claim 18, wherein the solid-phase surfacecomprises an anti-GPIbα antibody that binds to GPIbα or functionalfragment thereof
 20. The kit of claim 11, wherein the at least twomutations are selected from the group consisting of D235Y/G233V,D235Y/M239V, G233V/M239V and D235Y/G233V/M239V.